Human TROP-2 (Tacstd2, GA733-1 and EGP-1) (hereinafter also referred to as “hTROP-2”) is a single transmembrane, type 1 cell membrane protein consisting of 323 amino acid residues (see SEQ ID NO: 2), and this protein has been known to be overexpressed in various types of epidermal cell carcinomas. The presence of a cell membrane protein associated with immunological resistance, which is commonly expressed in both human trophoblasts and cancer cells, had been long suggested (Non-Patent Document 1). An antigen molecule recognized by mouse monoclonal antibodies (162-25.3, 162-46.2) reacting with the cell membrane protein of a human choriocarcinoma cell line BeWo was identified. This antigen molecule was considered as one of the molecules expressed in human trophoblasts, and was named as Trop-2 (Non-Patent Document 2). Thereafter, the same molecule was discovered by other researchers. That is to say, a tumor antigen recognized by a mouse monoclonal antibody GA733 which is obtained by immunization with stomach cancer cells SW948 was named as GA733-1 (Non-Patent Document 3), and an epithelial glycoprotein recognized by a mouse monoclonal antibody RS7-3G11 which is obtained by immunization with non-small cell lung cancer cells was named as an epithelial/carcinoma antigen, EGP-1 (Non-Patent Document 4). In 1995, the Trop-2 gene was cloned, and as a result, it was confirmed that these are the same molecules (Non-Patent Document 5). Moreover, it was clarified that the molecule has a function to amplify intracellular calcium signals in cancer cells (Non-Patent Document 6), and therefore, it is also referred to as a tumor-associated calcium signal transducer 2 (TACSTD2).
The hTROP-2 gene is mapped on chromosome 1p32, and it constitutes a TACSTD gene family together with GA733-2 having a homology of approximately 50% therewith (which has been known as “TACSTD1,” “epithelial glycoprotein EGP-2,” “EpCAM” or “Trop-1”) (Non-Patent Document 7). The hTROP-2 protein (323 amino acid residues; SEQ ID NO: 2) has a molecular weight of approximately 36K Dalton, and this protein consists of a hydrophilic signal peptide (15t to 26th amino acids), an extracellular domain (27th to 274th amino acids), a transmembrane domain (275th to 297th amino acids) and an intracellular domain (298th to 323rd amino acids). The extracellular domain has four heterogeneous N-linked glycosylation sites, and its apparent molecular weight is increased by 11 to 13K Dalton due to addition of sugar chains (Non-Patent Document 5). It is considered that TACSTD gene family has a characteristic thyroglobulin (TY) repeat sequence in the extracellular domain and is associated with the proliferation, invasion and metastasis of cancer cells.
To date, a physiological ligand of hTROP-2 has not been identified, and the molecular function thereof has not been clarified. However, it has been described that hTROP-2 transmits a calcium signal in tumor cells (Non-Patent Document 6). In addition, from the facts that intracellular serine 303 is phosphorylated by Ca2+-dependent protein kinase C (Non-Patent Document 4) and that hTROP-2 has a PIP2-binding sequence in its intracellular domain, it has been suggested that hTROP-2 has a signaling function in tumor cells (Non-Patent Document 8).
As a result of in vitro diagnostic studies such as immunohistochemistry (IHC) and flow cytometry analysis, overexpression of hTROP-2 in many types of epithelium-derived carcinomas such as stomach cancer, lung cancer, colon cancer, ovary cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer and esophagus cancer has been reported. In contrast, the expression of hTROP-2 in normal tissues is limited to cells in the epithelial region, and the expression level of hTROP-2 in normal cells is lower than that in cancer cells. Thus, the association of TROP-2 with tumor formation is suggested (Patent Documents 1-3 and 9).
Moreover, in the analysis of an hTROP-2 expression level used as a biomarker in clinical samples, it has been demonstrated that, when hTROP-2 is highly expressed, correlating with the malignancy of colon cancer (Non-Patent Documents 10 and 11), pancreatic cancer (Non-Patent Document 12) or oral cancer (Non-Patent Document 13), the possibility of metastasis or recurrence of such cancer is significantly high. Furthermore, in a large-scale gene expression analysis using a cDNA microarray technique, hTROP-2 has been identified as a gene cluster, which is overexpressed at the highest level in severe papillary carcinoma of the ovary, in comparison with in normal ovary epithelium (Non-Patent Document 14).
Still further, in recent years, an important role of hTROP-2 in tumor formation has been demonstrated in the models by using colon cancer cells (Non-Patent Document 15). Since the expression of hTROP-2 promotes the anchorage-independent cell proliferation of tumor cells and is required for the tumor formation and proliferation of cancer cells subcutaneously transplanted in immunodeficient mice, it raised the possibility that hTROP-2 would act as a functional tumor antigen and would be used as a new therapeutic target.
To date, studies regarding the anti-tumor effects of several anti-hTROP-2 antibodies have been reported. An RS7 antibody (Patent Document 1) has been examined by employing in vivo models, in which radiolabeled antibodies were used, and anti-tumor activity was demonstrated in nude mouse xenograft models. However, the anti-tumor effects by antibody alone (a naked antibody) have not been reported.
In addition, the cytotoxicity of a cytotoxin-attached anti-hTROP-2 monoclonal antibody BR110 (Patent Document 2) on human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981 in in vitro experiments has been reported. However, the cytotoxicity of a naked antibody or an immune conjugate of BR110 in vivo has not been disclosed.
In recent years, it has been reported that an isolated monoclonal antibody, which was produced from a hybridoma cell line AR47A6.4.2 or AR52A301.5 obtained by immunizing mice with human ovary cancer tissues, bound to hTROP-2, and that, for the first time, it exhibited, as a naked antibody, anti-tumor activity on nude mouse xenograft models, as well as cytotoxicity in vitro (Patent Documents 3 and 4). In these patent documents, the aforementioned antibody exhibited anti-tumor effects by treatment with antibody alone in mouse xenograft models, into which pancreatic cancer cell lines BxPC-3 and PL45, a prostate cancer cell line PC-3, a breast cancer cell line MCF-7 and a colon cancer cell line Colo205 had been transplanted. The therapeutic effects of the antibody have appeared in the models, into which BxPC-3 cells had been transplanted. Other than this, tumor formation and proliferation were only partially (approximately 40% to 60%) suppressed by the preventive administration of the antibody, and an extremely large amount (approximately 20 mg/kg) of the antibody was necessary for such suppression of tumor formation and proliferation.
Based on the above-mentioned previous findings, the potential use of the anti-hTROP-2 antibody as an anti-tumor antibody has been suggested. However, not all of the anti-hTROP-2 antibodies exhibit anti-tumor effects by treatment with antibody alone as naked antibodies in vivo. The antibodies exhibit different functions to hTROP-2, depending on a binding site, affinity and the profile of a monoclonal antibody.    Patent Document 1: U.S. Pat. No. 6,653,104    Patent Document 2: U.S. Pat. No. 5,840,854    Patent Document 3: U.S. Pat. No. 7,420,040    Patent Document 4: U.S. Pat. No. 7,420,041    Non-Patent Document 1: Faulk W P, et al., Proc. Natl. Acad. Sci. U.S.A., 75(4), pp. 1947-1951 (1978)    Non-Patent Document 2: Lipinski M, et al., Proc. Natl. Acad. Sci. U.S.A., 78(8), pp. 5147-5150 (1981)    Non-Patent Document 3: Linnenbach A J, et al., Proc. Natl. Acad. Sci. U.S.A., 86(1), pp. 27-31 (1989)    Non-Patent Document 4: Basu A, et al., Int. J. Cancer, 62(4), pp. 472-479 (1995)    Non-Patent Document 5: Fornaro M, et al., Int. J. Cancer, 62(5), pp. 610-618 (1995)    Non-Patent Document 6: Ripani E, et al., Int. J. Cancer, 76(5), pp. 671-676 (1998)    Non-Patent Document 7: Calabrese G, et al., Cell Genet., 92(1-2), pp. 164-165 (2001)    Non-Patent Document 8: El Sewedy T et al., Int. J. Cancer, 75(2), pp. 324-330 (1998)    Non-Patent Document 9: Cubas R, et al., Biochim. Biophys. Acta., 1796(2), pp. 309-314 (2009)    Non-Patent Document 10: Ohmachi T et al., Clin. Cancer Res., 12(10), pp. 3057-3063 (2006)    Non-Patent Document 11: Fang Y J, et al., Int. J. Colorectal Dis., 24(8), pp. 875-884 (2009)    Non-Patent Document 12: Fong D, et al., Br. J. Cancer, 99(8), pp. 1290-1295 (2008)    Non-Patent Document 13: Fong D, et al., Mod. Pathol., 21(2), pp. 186-191 (2008)    Non-Patent Document 14: Santin A D, et al., Int. J. Cancer, 112(1), pp. 14-25 (2004)    Non-Patent Document 15: Wang J, et al., Mol. Cancer Ther., 7(2), pp. 280-285 (2008)